Computer-implemented method and system for spectroscopic analysis of biological material

ABSTRACT

A computer-implemented method for spectroscopic analysis of biological material is provided that includes analyzing samples of biological material from a plurality of sources, and delivering samples of biological material to at least one flow cell for spectroscopy, and determining whether the spectroscopic analysis for each sample of the plurality of samples is or is predicted to be ambiguous in that it is affected by at least two non-discriminable factors. If such a determination is made, a disambiguating step can be performed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/042,837, filed Sep. 28, 2020, which is the U.S. National Stage ofInternational Application No. PCT/EP2019/057992, filed Mar. 29, 2019,which was published in English under PCT Article 21(2), which in turnclaims the benefit of European Patent Application No. 18 000 314.7,filed Mar. 29, 2018. The prior applications are incorporated herein byreference in their entirety.

FIELD

The following description relates to spectroscopic analysis performedon-line for a bioreactor. In particular, the description relates to acomputer-implemented method and system for spectroscopic analysis ofbiological material.

BACKGROUND

Industrial processes, particularly biopharmaceutical processes, as wellas research and development processes, increasingly involve the use ofbioreactors for cultivating microorganisms under controlled conditions.Processes taking place within bioreactors involve a plurality ofvariables linked by complex relations, so that bioreactor monitoring andcontrolling are particularly challenging.

SUMMARY

It is an object of the invention to provide an effective manner ofmonitoring a bioreactor by means of spectroscopy in order to increaseefficiency, productivity and quality of processes taking place therein.In particular, it is an object to provide an efficient calibration forspectroscopic analysis, as well as an insight into the root causes ofspectra deviation. It is a further object to facilitate thereproducibility of the process even at different scales.

The achievement of this object in accordance with the invention is setout in the independent claims. Further developments of the invention arethe subject matter of the dependent claims.

According to one aspect of the invention, a computer-implemented methodfor spectroscopic analysis of biological material is provided. Themethod includes an analysis step comprising:

taking, by a liquid router, a plurality of samples of biologicalmaterial from a plurality of sources, wherein each sample of theplurality of samples is taken from a source of the plurality of sources:delivering, by the liquid router, the plurality of samples of biologicalmaterial to at least one flow cell for spectroscopy;performing, by at least one spectrometer connected to the at least oneflow cell, the spectroscopic analysis of the plurality of samples ofbiological material;determining whether the spectroscopic analysis for each sample of theplurality of samples is or is predicted to be ambiguous in that it isaffected by at least two non-discriminable factors; if the spectroscopicanalysis for a given sample of the plurality of samples of biologicalmaterial is or is predicted to be ambiguous, performing a(post-analysis) disambiguating step comprising:

(a1) routing, by the liquid router, the given sample of biologicalmaterial from the at least one flow cell to a manipulation station:

(b1) manipulating, at the manipulation station, the given sample ofbiological material in order to impact the spectroscopic analysis;

(c1) delivering, by the liquid router, the given manipulated sample ofbiological material from the manipulation station to the at least oneflow cell;

(d1) performing, by the at least one spectrometer, the spectroscopicanalysis of the given manipulated sample of biological material.

The media for a bioreactor process include biological material, i.e.material comprising a biological system, such as cells, cell components,cell products, and other molecules, as well as material derived from abiological system, such as proteins, antibodies and growth factors.Further media may include chemical compounds and various substrates. Inthe following, “a sample of biological material” will be used to denotea sample containing at least some biological material, which may or maynot contain additional substances.

A spectroscopic analysis investigates how matter interacts with light inorder to obtain information on the matter, e.g. to determine thefunctional groups in organic compounds or chemical composition.Accordingly, a spectroscopic analysis of biological material may provideinformation on the biological material, such as cell density or glucoseconcentration. An advantage of spectroscopic analysis is that it is anon-destructive technique. An exemplary spectroscopic technique is theRaman spectroscopy, which uses a laser light source and can identifyboth organic and inorganic substances in various states such as liquid,solid, emulsion and so on. Other kinds of spectroscopy include e.g.ultraviolet-visible (UV-Vis), fluorescence, near-infrared (NIR) andscattering spectroscopy.

The method comprises taking, by a liquid router, a plurality of samplesof biological material from a plurality of sources of biologicalmaterial. The plurality of sources may comprise a plurality ofdisposable bioreactors, e.g. Sartorius products like Ambr®, UniVessel®and BIOSTAT STR® Exemplarily, the sources may be given by an Ambr®system, which comprises multiple single-use micro bioreactor vessels.

A bioreactor may be used for a variety of processes, such as industrialprocesses, particularly biopharmaceutical processes. Other examplesinclude research and development processes or scientific research. Inparticular, a bioreactor process may generally comprise convertinginputs or ingredients into a finished product.

Such a production process may involve chemical or microbiologicalconversion of material in conjunction with the transfer of mass, heat,and momentum. The process may include homogeneous or heterogeneouschemical and/or biochemical reactions. The process may comprise but isnot limited to mixing, filtration, purification, centrifugation and/orcell cultivation.

Possible products may include a transformed substrate, baker's yeast,lactic acid culture, lipase, invertase, rennet. Further exemplarybiopharmaceutical products that can be produced include the following:recombinant and non-recombinant proteins, vaccines, gene vectors, DNA.RNA, antibiotics, secondary metabolites, growth factors, cells for celltherapy or regenerative medicine, half-synthesized products (e.g.artificial organs).

Generally, a process occurring in a bioreactor is governed by aplurality of process parameters. e.g. temperature and stirring speed.The values of these process parameters may be specifically adjustedprior to and/or during performing the process, e.g. they can be set byan operator or by a control system. The process may be described by aseries of steps.

Exemplarily, the plurality of sources may partially or in the totalitybe used to obtain the same product by performing variations of the sameproduction process. In particular, the production process for a givenproduct may be thought of as comprising some essential steps to beperformed with some essential inputs, while the process parameters areconstrained within boundaries determined by scientific principles and/orfeasibility.

Variations of the production process may, thus, be given by one or moreof the following: different values of one or more process parameters,additional optional steps, additional optional inputs. By way of furtherexample, process variation may be produced by:

-   -   variation in culture organism or clone;    -   variation in base medium;    -   changes in inoculum density or volume;    -   changes in set-points e.g. temperature, pH, or profiles e.g. of        temperature    -   changes in feeding regime, either in terms of feed profile, or        in terms of feedback control;    -   changes in culture duration.

Accordingly, a variation of a given production process may be performedin each source, so that the plurality of sources correspond to arespective plurality of process variations. As an example the set pointfor pH might be 7.2, wherein the accuracy of the pH measurement is+−0.05. Process variations with a setpoint of 7.15 and 7.25 may beperformed to cover the normal occurring variation.

Further, complex processes such as those taking place in bioreactorspresent intrinsic variations even if the same steps with the sameprocess parameters are performed. This partly arises from the non-linearamplification of small variations between otherwise highly similarculture vessels or treatments. Accordingly, two or more sources may beused for nominally identical processes, which in reality are alsovariations of the same process due to intrinsic variations.

Each sample of the plurality of samples is taken from a source of theplurality of sources. In particular, in the context of this application,a sample may be a portion of biological material characterized by itsorigin, in that samples from different sources. e.g. bioreactors, areconsidered distinct samples, irrespectively of the specific biologicalmaterial which constitutes the sample. In some examples, the biologicalmaterial in two samples may be the same in the sense that it comprisesthe same elements, e.g. same cells and same nutrients. However, sincethe two samples are extracted from two bioreactors performing twodifferent process variations (due to extrinsic or intrinsic variations),the samples are regarded as distinct. The samples of biological materialare in liquid form although they may have particulates (cellular orother) in suspension.

A liquid router takes the plurality of samples of biological materialfrom the plurality of sources. The liquid router is configured to handleliquid material. e.g. the biological material, by retrieving it, routingit and delivering it. Exemplarily, the liquids handled by the liquidrouter may be divided into three categories: biological materials,cleaning liquids and standard reference liquids. Standard referenceliquids may include:

-   -   analytes of interest. e.g. glucose, lactate, protein, product,        viable or dead cells, diluted in water, buffer or media:    -   mixes of the above, according to a Design of Experiments        approach, to reduce model error from a limited number of        samples;    -   water, buffer or media.

Accordingly, the liquid router may comprise means for drawing offliquid, e.g. from the bioreactor or a plate well or any other liquidholder, and for dispensing liquid, e.g. to a plate well or into a wastestation, such as a pipettes or syringes. Further, the liquid router maycomprise means for conveying liquid from a first location to a second,different location, such as lines in connection with pumps and/ormovable components such as a robotic arm. Exemplarily, the liquid routermay comprise a liquid handling robot that performs automated pipetting,and the pumped lines may also be automatically controlled.

In particular, the liquid router may connect different components of asystem for monitoring the bioreactors by means of an on-line analysis.Indeed, monitoring methods for process setups, such as bioreactors, andthe analysers used therefore are usually classified into fourcategories: on-line, in-line, at-line and off-line. On-line and in-lineanalyses both involve a real-time, automated monitoring. In-lineanalyses are performed by analysers (sensors or probes or any othermeasuring devices) that are placed in direct connection with thebioreactor (e.g. located within or in close proximity to the bioreactor)and measure parameters (e.g. pH) that can be directly measured. On-lineanalyses may be considered enhanced in-line analyses comprisingelaborating the measurements to interpret them and derive information.Conversely, at-line and off-line analyses involve manual intervention ofoperators and delayed analysis results. At-line analysers may be in ageneral proximity to the bioreactor, so that an operator can easilycollect samples from the bioreactor and feed them to the at-lineanalyser. Off-line analysers may be located remotely from the bioreactorand may offer a large variety of tests at the cost of a long turn-aroundtime.

The proposed method provides an on-line ex-situ analysis, i.e. thesamples are removed from the bioreactors to be analysed. Taking theplurality of samples of biological material from the plurality ofsources may be performed sequentially, i.e. one sample is taken afterthe other, or simultaneously, i.e. all samples are taken at the sametime, or a combination of both, in the sense that groups of samples maybe taken simultaneously and each group after the other sequentially.Exemplarily, the plurality of samples of biological material are takensequentially.

In particular, the liquid router delivers the plurality of samples ofbiological material to at least one on-line ex-situ flow cell forspectroscopy. Exemplarily, each time a sample is taken it may betransferred from the source to a sample cup before being delivered tothe flow cell. The flow cell (also called “measurement cell”) comprisesa body including at least one inlet through which a liquid can beintroduced, at least one outlet through which the liquid can be removedand at least one optical window. An optical window is an element (e.g. aplate) that is at least partly transparent, i.e. allows at least partlypassage of electromagnetic radiation in a wavelength region of interestfor the spectroscopy. e.g. 190-2500 nm. Exemplarily, the electromagneticradiation may be visible, ultraviolet and/or infrared light. The atleast one optical window may comprise two plane-parallel windows of atleast partially optical transmittant material (transmittance>20%) in therelevant wavelength range, e.g. 190-2500 nm. The path length between thetwo plane-parallel windows may be between about 10 μm and about 1 cm, inparticular between about 0.1 mm and about 2 mm. An exemplary volume of acavity in between the plane-parallel windows may be much less than 1 ml,especially between about 20 and about 200 μl.

Exemplarily, the flow cell may be designed with straight inlet andoutlet in order to reduce the likelihood of clogging and it may becomposed by two or more detachable parts in order to allow easy accessfor cleaning. The inlet and the outlet may form two channels on oppositesides of the flow cells that can be connected to tubes and may, thus,represent a bottom opening and a top opening. The diameter of thechannels may e.g. be between about 0.1 and about 10 mm, in particularbetween about 0.1 mm and about 1.5 mm. In some examples, at least one ofthe inlet and outlet may be used both for introducing and removing theliquid.

Exemplarily, the flow cell may be temperature controlled, e.g. by meansof a temperature sensor in combination with hot and/or cold fluids (suchas via a heat exchanger, a bath circulator, etc.), at least one Peltierelement and/or one or more insulation measures. Temperature control inthe flow cell is advantageous, in that it ensures consistency betweensample conditions and, consequently, of the resulting spectral features.In particular, a temperature-controlled flow cell spectroscopy mayprovide results that allow a more direct comparison with resultsobtained from in-vessel measurements. Indeed, in-vessel measurements arealways temperature-controlled. If the flow cell measurements have to becombined with in-vessel measurements, it might be beneficial to have thesame temperature in both systems and, thus, to have atemperature-controlled flow cell. Accordingly, the spectroscopic resultsare more suitable for a comparison to results from inline spectroscopyof other bioreactors and, therefore, data can be combined and/orcompared more easily.

The flow cell may allow for simultaneous detection of severalmeasurement modes (transmission, reflection, transflection) and variousoptical spectroscopies (UV-Vis, NIR, fluorescence, Raman, scattering).In particular, the applied technique is independent of flow cell designbut only depends on fibre design and connected spectrometer/lightsources.

The flow cell may be configured to be connected to a probe comprising anoptical fibre. In particular, the flow cell may be connected to a fibreprobe head including beam shaping optics and, via the probe head, tofibres connected to the probe head. Alternatively, the beam shapingoptics may be integrated into the fibres and the probe head may onlyfixate the fibres to ensure optimal fibre positioning with regard to theflow cell. The fibre head and the flow cell may be detachably connected.e.g. by clipping. The fibre head may be either directly connected tofibres (e.g. the fibres may be glued to fibre head) or fibres may bescrewed into fibre head via a coupling (e.g. via SMA coupling).Exemplarily, there may be no fibre-fibre connection inside the probe.The light of the fibre is guided by free optics into the flow cellcavity (and back).

The beam shaping optics may be contained in the probe head, while theflow cell contains no such optics. In particular, in one example, thefibre head may also enable adjustment of the focal point. Exemplarily,the probe head may be a reusable part. The flow cell may be reusable orit may also be single use. The single-use flow cell body may be made ofa polymer and can be manufactured by injection molding or rapidprototyping (e.g. laser sintering or fused deposition modeling).

The liquid router may deliver the plurality of samples of biologicalmaterial sequentially to the at least one flow cell, so that theplurality of samples of biological material are analysed one after theother. When handling the plurality of samples of biological material,the liquid router may take cleaning liquid from a cleaning liquidstation to cleanse itself, the one or more flow cells and other systemcomponents in order to avoid contamination.

In some examples, the liquid router may deliver the at least one sampleof biological material to a plurality of flow cells. The plurality offlow cells might address different modes of spectroscopy, e.g. indifferent wavelength regions, or different optical paths e.g. absorbanceversus transmission. Different types of spectroscopy may provideorthogonal information on a sample. Exemplarily, different types ofspectroscopy may be used in combination to resolve analytes that wouldotherwise be indistinguishable.

Exemplarily, the liquid router may deliver each sample of biologicalmaterial sequentially to the plurality of flow cells, namely route agiven sample of biological material to a first flow cell and, after afirst spectroscopic analysis, deliver the given sample of biologicalmaterial from the first flow cell to a second flow cell and, possibly,from the second flow cell to a third flow cell and so on.

Alternatively a given sample of biological material may be divided bythe liquid router into two or more subsamples prior to delivery to theflow cells. Accordingly, the liquid router may deliver the given sampleof biological material as split into two or more subsamples to theplurality of flow cells by routing a subsample to each flow cell. Inother examples, the liquid router may perform both sequential deliveryand splitting of the at least one sample of biological material, e.g.delivering the given sample of biological material to a first flow celland, after the first spectroscopic analysis, splitting the sample intotwo subsamples, one of which is delivered to a second flow cell whilethe other is delivered to a third flow cell.

In particular, delivery to a given flow cell may modify the sample, so asecond spectroscopic analysis in a second flow cell has the potential toquantify any modification of the sample.

Each sample of the plurality of samples of biological material may bedelivered to all or some or only one of the plurality of flow cells.Exemplarily, all samples may be delivered to all flow cells. This may bedone sequentially, in that a first sample is delivered to all flow cells(sequentially or in form of subsamples, as described above), then asecond sample is delivered to all flow cells and so on, orsimultaneously, e.g. a first sample is delivered to a first flow cellwhile a second sample is delivered to a second flow cell and then thesecond sample is delivered to the first flow cell while the first sampleis delivered to the second flow cell. In other examples, the deliverymay be partly sequential and partly simultaneous.

A method for delivering a sample to a flow cell may ensure that thesample is substantially bubble-free and, therefore, ensure acquisitionof a representative sample, by reducing and/or eliminating the risk ofspectral artefacts caused by gas at a gas/liquid interface. Exemplarily,one such method may be based on one or more of the followingarrangements.

The flow cell may be positioned such that gravity supports filling theflow cell and/or emptying the flow cell through the inlet and/or outlet,thereby preventing air from being trapped within the flow cell, inparticular between or adjacent to the one or more windows or transparentsubstrates through which the spectroscopic measurement is performed. Inother words, the position of the flow cell may be non-horizontal,meaning that a longitudinal axis extending through the inlet/outletchannel(s) (i.e. an axis corresponding to the macroscopic direction ofthe fluid when it enters/leaves the flow cell) is at an angle differentfrom 0° or 180° with reference to an axis parallel to the ground. If theinlet/outlet channel is approximately a cylindrical surface, thelongitudinal axis may have the direction (i.e. be parallel to) of thegeneratrix of the cylindrical surface. The longitudinal axis may bepositioned at an angle of about 30° to about 150°, in particularapproximately in the range of about 75°-105° relating to ground level.Exemplarily, the flow cell may be positioned vertically. i.e. at anangle of about 90°.

As mentioned above, only one opening out of the two openings, the inletand the outlet, may be used for the liquid and it may be substantiallylocated at the bottom of the flow cell, i.e. at the surface of the flowcell closer to the ground when the flow cell is located in the automatedsystem. In other words, the flow cell may be positioned or oriented sothat its liquid opening is situated substantially facing the ground.Accordingly, the liquid of the sample may be delivered through thebottom opening when filling the flow cell, while air may be released viathe other opening. e.g. a top opening. Then, the flow cell may beemptied via the bottom opening, which was also used for filling the flowcell, and while the flow cell gets emptied of the liquid, air may enterthe flow cell via the top opening.

The at least one flow cell is connected to at least one spectrometer,e.g. via one or more optical fibres that guide electromagnetic radiationfrom the flow cell to the spectrometer. In particular, in the case of aplurality of flow cells, each flow cell is connected to onespectrometer. Indeed, for spectroscopic analysis, an electromagneticradiation source, such as a laser, emitting in the wavelength region ofinterest is used to irradiate the content of the flow cell via the atleast one optical window. The electromagnetic radiation afterinteraction with the biological material of the sample in the flow cell(e.g. scattering, absorption etc.) is guided via the optical fibre tothe spectrometer. The spectrum of this electromagnetic radiation is thenanalysed by the spectrometer, which accordingly performs thespectroscopic analysis.

After the spectroscopic analysis for a single sample is completed, theliquid router may deliver the sample of biological material from theflow cell or one of the flow cells to a waste station for disposal.Afterwards, the liquid router may clean itself and the flow cell(s) withcleaning liquid and proceed to analyse the subsequent sample of theplurality of samples of biological material. The liquid router maycomprise non-communicating parts that can be washed independently fromeach other. Accordingly, analysis steps for subsequent samples maypartly overlap in order to increase throughput, e.g. while a firstsample is in the flow cell being analysed, a second sample may be takenfrom a bioreactor. However same actions may be performed onlysequentially, e.g. taking a first sample may not overlap with taking asecond sample, and analysing a first sample may not overlap withanalysing a second sample.

To summarize what explained so far, the method comprises an analysisstep that, in the simplest example, may comprise taking, by the liquidrouter, a plurality of samples of biological material from a pluralitysources and delivering them to at least one flow cell for spectroscopicanalysis, which is performed by a spectrometer, and then discarding thesample in a waste station. Further, the method may comprise performingthe plurality of process variations in the plurality of sources. Asexplained below, the plurality of process variations may be used tocalibrate a spectroscopy model used for the spectroscopy analysis.

The spectroscopic analysis may provide one or more results, wherein aresult is a piece of information about the analysed sample, such as aquantitative and/or qualitative determination of the content of thesample, e.g. an estimate of the properties of analytes in the sample.For example, cell concentration or glucose concentration may bedetermined. The information about a sample provided by the spectroscopicanalysis can be used for monitoring the evolution and performance of theproduction process in the bioreactor from which the sample is taken.

However, spectroscopy is an indirect analytical method that requires amodel to link the spectral features to results. Accordingly, a result isinferred in real time from features of the electromagnetic radiationspectrum on the basis of the model (on-line analysis). In the majorityof cases, the spectroscopic analysis may be ambiguous in that it may beaffected by two or more non-discriminable factors. Non-discriminablefactors have the same effect on the spectrum according to the employedmodel, wherein the term “same” particularly should be interpreted asnon-distinguishable within the given sensitivity and model accuracy. Inother words, the non-discriminable factors are strongly correlated andeasily confounded on the basis of the model.

In particular, the interpretation of the feature(s) of the spectrum fromwhich a result is obtained, such as the strength of a spectral line, mayfrequently be affected by the at least two non-discriminable factors.Accordingly, the spectroscopic analysis may be ambiguous in the sensethat it gives different results according to which factor has actuallydetermined the given feature. In other words, the result may be e.g. apiece of information A or a piece of information B but the model usedfor the spectroscopic analysis cannot determine whether it is A or B.

In production processes occurring in bioreactors, such as cellcultivation, changes in one analyte are often correlated with those of asecond analyte, due to mechanisms within the culture e.g. consumption ofglucose by cells with concomitant production of lactate. Consequently, amodel built just on samples from culture cannot typically discriminateglucose and lactate, because both are correlated with time into theprocess; similarly, a model built just on samples from culture cannottypically discriminate titer and viable cell density (VCD), becauseincreases in titer and VCD are highly temporally correlated.

If the spectroscopic analysis is or suspected to be ambiguous, adisambiguating step may be performed. Conversely, if there is noambiguity, the disambiguating step is not performed. The purpose of thedisambiguating step is breaking the correlation between the at least twonon-discriminable factors. It should be noted that the non-discriminablefactors may comprise only factors of interest, i.e. whosefeatures/properties (e.g. concentration) are desired to be obtained viathe spectroscopic analysis, or both factors of interest and confoundingfactors whose features/properties are not to be obtained via thespectroscopic analysis, because they are not relevant or they can beobtained by an alternative technique in a more precise manner. Forexample, two non-discriminable factors may comprise two factors ofinterest such as glucose and lactate, or one factor of interest,glucose, and a confounding factor such as cell debris or temperature.Confounding factors may include but are not limited to temperature, pH,fluorescence, scattering, spectral background, process time.

The determination of whether the spectroscopic analysis is (or issuspected to be) ambiguous may be done on the basis of predictionsand/or on the basis of the actual spectroscopic analysis.

In particular, the analysis step may comprise determining, prior toperforming the spectroscopic analysis, whether the spectroscopicanalysis is predicted to be ambiguous, as well as determining, afterperforming the spectroscopic analysis, whether the spectroscopicanalysis was indeed ambiguous. Actually, the step of determining whetherthe spectroscopic analysis for each sample is predicted to be ambiguousmay be performed at any moment with respect to the spectroscopicanalysis, since it is not bound to its results, namely before or afterthe spectroscopic analysis. In particular, it may be performed prior tothe spectroscopic analysis, e.g. prior to taking the plurality ofsamples.

If no ambiguity is predicted or detected, the analysis step may reduceto the simplest example discussed above. If an ambiguity is predictedbefore the spectroscopic analysis is performed or identified after thespectroscopic analysis has been performed, the sample may undergo adisambiguating step. The disambiguating step may take place after thespectroscopic analysis and may be referred to in the following as“post-analysis” disambiguating step. After the post-analysisdisambiguating step, the sample is returned to the at least one flowcell for a second spectroscopic analysis. If the ambiguity is predicted,a disambiguating step may also be performed prior to delivering a sampleto the at least one flow cell for a first spectroscopic analysis. Insome examples, both a pre-analysis disambiguating step and apost-analysis disambiguating step may be performed.

The spectroscopic analysis may be predicted to be ambiguous e.g. on thebasis of theoretical modelling and/or experimental data. In particular,the spectroscopic analysis may be predicted to be ambiguous based on(the particular variant of) the production process occurring in thesource from which the sample is taken. For example, the determinationstep may amount to providing a set of instructions for automaticexecution of the analysis step which includes the disambiguating stepfrom the outset. Alternatively, there may be one or more control flagsassociated with the production process in general or with particularcharacteristics of the process in a given source, wherein a control flagindicates a possible or certain ambiguity. E.g., the flag may be 0 ifthe risk of ambiguity is predicted to be null or negligible (such aslower than a specific, predetermined or predeterminable threshold) and 1if the risk of ambiguity is substantial (such as higher than a specific,predetermined or predeterminable threshold). If at least one controlflag indicates ambiguity, the disambiguating step particularly isperformed. The one or more control flags related to the process may be(automatically) checked at any moment prior to the spectroscopicanalysis, as well as after the spectroscopic analysis. When thespectroscopic analysis is predicted to be ambiguous, the disambiguatingstep may be a pre-analysis disambiguating step or a post-analysisdisambiguating step. In a particular example, it is a post-analysisdisambiguating step.

The spectroscopic analysis may be found to be ambiguous after it hasbeen performed. i.e. on the basis of the actual spectroscopic analysis.Exemplarily, the one or more results of the spectroscopic analysis maybe provided in such a way that ambiguity is indicated (e.g. presence ofspecific symbols such as ‘?’ or low levels of confidence for certainresults). Based on the indication in the results, the disambiguatingstep may be performed. When the spectroscopic analysis is found to beambiguous, the disambiguating step is a post-analysis disambiguatingstep.

While, when the ambiguity is predicted, the nature of the disambiguatingstep may be fixed, in the sense that is determined a priori how thesample is to be manipulated, determining the ambiguity on the basis ofthe actual results may allow for a modulation “on the fly” of themanipulation procedure or even of the production process to which thesample refers to.

Generally, the disambiguating step may comprise routing the sample ofbiological material for which an ambiguity has been determined(predicted or identified) to a manipulation station. In a pre-analysisdisambiguating step, the sample of biological material may betransferred from the source to the manipulation station. In apost-analysis disambiguating step, the liquid router may transfer thesample from the flow cell back to the sample cup and from there to themanipulation station. Accordingly, a post-analysis disambiguating stepmay comprise: routing, by the liquid router, the given sample ofbiological material from the at least one flow cell to a manipulationstation; manipulating, at the manipulation station, the given sample ofbiological material in order to impact the spectroscopic analysis;delivering, by the liquid router, the given manipulated sample ofbiological material from the manipulation station to the at least oneflow cell; performing, by the at least one spectrometer, thespectroscopic analysis of the given manipulated sample of biologicalmaterial. A pre-analysis disambiguating step may comprise: routing, bythe liquid router, the given sample of biological material from thesource to the manipulation station; manipulating, at the manipulationstation, the given sample of biological material in order to impact thespectroscopic analysis; delivering, by the liquid router, the givenmanipulated sample of biological material from the manipulation stationto at least one flow cell; performing, by at least one spectrometerconnected to the at least one flow cell, the spectroscopic analysis ofthe given manipulated sample of biological material.

Exemplarily, the disambiguating step may be performed on a differentsample from the same source of biological material of the sample forwhich an ambiguity is determined (predicted or identified). Accordingly,after determining whether the spectroscopic analysis for a sample is oris predicted to be ambiguous, the disambiguating step may comprise oneor more of the following:

-   -   (a2) taking, by the liquid router, a secondary sample from the        same source of the given sample for which the spectroscopic        analysis is or is predicted to be ambiguous:    -   (b2) routing, by the liquid router, the secondary sample of        biological material from the source to a manipulation station;    -   (c2) manipulating, at the manipulation station, the secondary        sample of biological material in order to impact the        spectroscopic analysis;    -   (d2) delivering, by the liquid router, the manipulated secondary        sample of biological material from the manipulation station to        the at least one flow cell; and    -   (e2) performing, by the at least one spectrometer, the        spectroscopic analysis of the manipulated secondary sample of        biological material.

Thus, the secondary sample is or may be taken for the purpose ofdisambiguation and may be directly transferred by the liquid router fromthe source of biological material to a manipulation station withoutperforming a spectroscopy measurement first. As explained above, asample is characterized by its origin, so that two samples from the samebioreactor can be considered equally “ambiguous” in terms of aspectroscopic analysis. The information obtained via the disambiguatingstep with the secondary sample may then be used for the spectroscopicanalysis of the original sample. The use of a secondary sample may beadvantageous, in that the secondary sample is out of the bioreactor fora shorter amount of time in comparison to a sample that has alreadyundergone a spectroscopic analysis, and thus has aged less. It may alsoreduce/eliminate possible modifications of the sample due to deliverythrough the system. Further, using a secondary sample simplifies theliquid handling and the automation of the process.

The manipulation station may be a location within the system at which atleast one liquid container for holding the sample during themanipulation is found and means for the manipulation are employed. Inparticular, means for the manipulation may comprise tools, e.g.heating/cooling device for modifying temperature, and/or substances,such as water for diluting or an acid for modifying the pH. The meansfor manipulation may be located at the manipulation station or may bemoved thereto, e.g. the substances may be stored at a storage stationand be routed by the liquid router to the manipulation station. Theliquid router itself may at least partly perform manipulation of thesample.

Generally, at the manipulation station, the sample may undergo physical,chemical and/or biological modifications. As discussed below, a sampleof biological material may be manipulated at a manipulation station fordifferent purposes. During the disambiguating step, the aim of themanipulation is to impact the spectroscopic analysis in order to try andbreak the correlation between the at least two non-discriminablefactors. In some cases, the manipulation leads to a modification of thespectrum. In other cases, the manipulation has no effect on thespectrum. Both the presence and the absence of an effect may help breakthe correlation. As already mentioned, the correlation to be broken maybe between factors of interest or factors of interest and confoundingfactors.

Exemplarily, manipulating the sample may comprise mixing the sample witha disambiguating substance, which is also known as “spiking”. The liquidrouter may draw the disambiguating substance from a source thereof andinject it into the at least one liquid container. The disambiguatingsubstance may or may not correspond to one of the non-discriminablefactors. For example, if the analyte of interest is lactate, the samplemay be spiked with glucose or lactate. Other examples include spikingwith water, with cells, with cell debris and/or with ions.

It is possible for there to be more than one component which gives riseto the need for disambiguation, and this multi-component disambiguationmay be achieved using a mixture of disambiguating substances.

In a particular example, manipulating may further comprise dividing thegiven sample of biological material in a plurality of subsamples andeach subsample is mixed with a different amount of the disambiguatingsubstance and/or multiple disambiguating substances. Accordingly,delivering the given manipulated sample may comprise delivering theplurality of manipulated subsamples; and performing the spectroscopicanalysis of the given manipulated sample may comprise performing thespectroscopic analysis of the plurality of manipulated subsamples.

Exemplarily, the at least one liquid container of the manipulationstation may comprise a plate with a plurality of wells. In particular,at the manipulation station, the sample of biological material may bedivided by the liquid router into a plurality of subsamples or aliquots,one contained in each well. The liquid router may also deliver one ormore substances for spiking the wells, wherein this step may take placebefore or after the subsamples are placed into the wells. In particular,each subsample may be differently manipulated, e.g. using differentamounts of the disambiguating substance or substances.

Accordingly, a result of the manipulation may be a plurality ofdifferently manipulated subsamples, which may be sent sequentially tothe flow cell for spectroscopic analysis. Each manipulated subsample maybe analysed and the information obtained from each spectroscopicanalysis may be used to break the correlation.

Manipulations other than spiking may include e.g. warming or cooling thesample, removing cell or cell debris from the sample, changing pH of thesample and/or changing ionic strength of the samples.

If there is a plurality of flow cells, and the spectroscopic analysisfor more than one flow cell is or is predicted to be ambiguous, adisambiguating step may be performed for each flow cell. Further,results from one flow cell may be used to disambiguate results obtainedfrom another flow cell.

To summarize, at least one disambiguating step may be performed for eachsample of the plurality of samples of biological material whosespectroscopic analysis is or is predicted to be ambiguous. It should benoted that the described manipulation of the sample for impacting thespectroscopic analysis may have a two-fold purpose: on one hand itallows to provide a result about the analysed sample and on the otherhand it allows a calibration of the model. Indeed, since a controlledmanipulation of the sample is performed, the sensitivity of the spectralcomponent(s) to a plurality of factors can be accurately studied. Inother words, the calibration of the model in which certain properties ofthe sample are identified as causes of certain features in the spectrumis made possible.

In view of the plurality of sources with process variations and thecountless possibilities for manipulating the analytes in the samples, anaccurate and refined calibration is possible, in which spectraldeviations can be linked to process/analyte changes. The processvariations carried out in the source of biological material and themanipulation of the samples may be organically considered as part of aprocedure for automatic calibration for spectroscopic analysis.Accordingly, the method for spectroscopic analysis is both a method forperforming spectroscopic analysis based on a model and obtaininformation about a sample and a method for calibrating a model used inthe spectroscopic analysis. In particular, in some cases, themanipulation of the sample and the process variations may serve more thefunction of introducing confounding factors to ensure model robustness,than discriminating two analytes of interest, as mentioned above.

As mentioned previously, even in a case in which no disambiguating stepis performed, the fact that a plurality of process variations is carriedout in a plurality of sources of biological material may alone provide acalibration of the model for spectroscopic analysis.

In certain circumstances, e.g. if there are more than twonon-discriminable factors, the disambiguating step may be repeated oneor more times. Accordingly, the sample may move from the flow cell tothe manipulation station and back to the flow cell, to be then broughtagain to the manipulation station and once again to the flow cell, andso on.

In a particular example, at least one of the plurality of samples ofbiological material may undergo pre-processing prior to thespectroscopic analysis. Accordingly, the analysis step may furthercomprise:

pre-processing at least one sample of the plurality of samples ofbiological material prior to performing the spectroscopic analysis, inparticular by at least one of: filtering, heating, normalizing pH,modifying ionic strength, diluting, metabolic inactivation.

The pre-processing is none other than a form of manipulation of thesample as described above. Accordingly, sample pre-processing mayinclude manipulating the at least one sample of biological material inorder to physically, chemically and/or biologically alter it. Unlike inthe disambiguating step, the aim of the manipulation in this case ispreparing and/or stabilizing the sample for spectroscopic analysis.Preparation of a sample for analysis might involve the physical removalof otherwise confounding factors. Stabilization of a sample for analysismight involve the inactivation of sample change, to prevent or diminishchange in the sample whilst spectral data are being captured.

For example, sample pre-processing may comprise but is not limited to:filtering or settling to remove particulates, modifying concentration(e.g. of proteins), metabolic inactivation, mixing, cooling, heating,normalizing pH, modifying ionic strength, diluting or any combinationthereof. Filtering or settling may be used to distinguish spectra withand without cells and therefore reflect the spectral properties of themedia relative to that of the cells and the media. Metabolicinactivation may be used to diminish sample change over time,particularly in the case of long (e.g. 15 min) spectral collection.

As already mentioned, sample pre-processing and sample manipulation fordisambiguation may involve, at least partly, the same procedures. Forexample, diluting the sample with water may be done for pre-processingand for disambiguation. Further, in some examples, sample pre-processingmay also comprise dividing the sample into a plurality of subsamples, asdescribed above. In particular, a plate with wells may also be used forpre-processing.

When a pre-analysis disambiguating step is performed, the pre-processingmay take place before, after or together with the pre-analysisdisambiguating step. The pre-processing may occur at the samemanipulation station used for the disambiguating step or at a differentmanipulation station. In any case, the liquid router may first route theat least one sample of biological material to the one or moremanipulation stations for pre-processing and pre-analysis disambiguatingstep and then route the at least one sample of biological material fromthe (last) manipulation station to the flow cell.

In another particular example, in addition to the on-line spectroscopicanalysis, a reference analysis, specifically an off-line, at- or on-linereference analysis may be performed, which provides a means of measuringthe analytes of interest, for which the spectroscopic analysis providesmodel-based estimates. Exemplarily, more than one reference analysis maybe performed.

In the case of at- or off-line reference analysis, the method mayfurther comprise: taking, by the liquid router, a subsample of at leastone sample of the plurality of samples of biological material;delivering, by the liquid router, the subsample to a transfer containerfor transfer to an at- or off-line reference system; performing, by theoff-line reference system, an at- or off-line reference analysis of thesubsample; comparing at least one result of the spectroscopic analysisby the spectrometer for the at least one sample of biological materialwith at least one result of the at- or off-line reference analysis. Inthe following, an off-line reference system is discussed, but the sameprinciples apply for an at-line reference system.

The liquid router may take a subsample out of a sample of biologicalmaterial, wherein the subsample is then transported to the off-linereference system and the rest of the sample is routed to the at leastone flow cell. Said in different terms, a sample of biological materialmay be split into two subsamples, one of which is delivered to the flowcell(s) while the other is sent to the off-line reference system. Inparticular, the sample of biological material may be delivered to atransfer container for transfer to the off-line reference system and thetransfer container may be manually brought to the off-line referencesystem.

The delivery to the transfer container may occur only before, only afteror before and after delivery to the at least one flow cell. If thedelivery occurs before, the sample of biological material may betransported by the liquid router from one source to the container fortransfer and then an aliquot may be taken from the container fordelivery to the at least one flow cell. If the delivery occurs after,the sample of biological material may be transported by the liquidrouter back from the flow cell to the sample cup and then to thetransfer container. In this way, the state of the (sub)sample forspectroscopic analysis is as similar as possible to the state of the(sub)sample used for the reference analysis. Accordingly, the timing ofthe reference analysis may be coordinated with the timing of thespectroscopic analysis, so that they are performed simultaneously. Inthis case, the result(s) of the spectroscopic analysis and the result(s)of the off-line reference analysis may be directly compared.

If the delivery to the transfer container occurs before and afterdelivery to the at least one flow cell, it may be possible to evaluateand account for a potential temporal change in the biological material.In particular, a first off-line reference analysis may be performed at apoint in time antecedent to the point in time at which the spectroscopicanalysis is performed and a second off-line reference analysis may beperformed at a point in time following the point in time at which thespectroscopic analysis is performed. The result(s) of the first off-linereference analysis and the result(s) of the second off-line referenceanalysis may be interpolated to obtain corresponding information at thetime point of the spectroscopic analysis, which can then be compared tothe result(s) thereof.

If a pre-analysis disambiguating step or post-analysis disambiguatingstep is performed, the subsample sent to the off-line reference systemmay be taken from the manipulated sample or from the non-manipulatedsample. In the latter case, corrections for the manipulation may be madeto the results of the off-line reference analysis.

The off-line reference system may be a measuring device configured tomeasure at least one property of at least one analyte of interest e.g.YSI glucose analyser, Nova Flex II, Cedex Cell Counter, ViCell CellCounter, an HPLC system, or a flow cytometer.

The measurement obtained thanks to the off-line reference system for agiven property (e.g. glucose concentration) may be compared to theestimate for that same property provided by the spectroscopic analysisby a computing device. This may be critical to calibrate the on-lineanalysis of the flow cell plus spectrometer. In particular, the modelused in the spectroscopic analysis may be adjusted so that there is amatch between the result(s) of the off-line reference analysis and ofthe spectroscopic analysis.

In some examples, a plurality of off-line reference systems may be usedfor measuring different properties. Accordingly, a plurality ofsubsamples may be drawn for the plurality of off-line reference systems.

In the case of an on-line reference analysis, the method may furthercomprise: taking, by the liquid router, a subsample of at least onesample of the plurality of samples of biological material; delivering,by the liquid router, the subsample to an on-line reference system;performing, by the on-line reference system, an on-line referenceanalysis of the subsample; comparing at least one result of thespectroscopic analysis by the spectrometer for the at least one sampleof biological material with at least one result of the on-line referenceanalysis. Accordingly, the only difference with an off-line referencesystem is that the liquid router may deliver the subsample directly tothe on-line reference system instead of the transfer container. Thus,what discussed above with regard to the off-line reference analysisapplies mutatis mutandis for an on-line reference system, in particularthe reference analysis may be performed prior to, at the same time asand/or after the spectroscopic analysis.

In a particular example, the analysis step may be further performed oneor more additional times at different time points, and the method mayfurther comprise determining a time-based profile of results of thespectroscopic analysis. The time-based profile may also be referred toas “trajectory”.

In particular, the results of the spectroscopic analysis may provideinformation on process parameters and/or analytes properties at thedifferent times during the evolution of the process. Accordingly, aplurality of trajectories may be obtained corresponding to a pluralityof process parameters/analyte properties as evolved when performing theprocess at a given scale. Exemplarily, the plurality of sources mayoperate at a small scale. A scale particularly refers to aconfiguration, e.g. a size of a setup used for executing the productionprocess, wherein the configuration determines, among others, thethroughput and the costs of the production process. Exemplarily, for aproduction process executed with a bioreactor, the scale value may referto the volume of the bioreactors, wherein a small scale in this case maybe 15 mL or 250 mL.

Each trajectory may be implemented as a curve or graph that describesthe time evolution of a quantity during the execution of the productionprocess. The one or more trajectories may be used when scaling up theproduction process. For example, the trajectories may be transferred toa larger scale (e.g. 2 L, 50 L, 200 L or 2000 L) and used forcomparison. In this case, an optically similar spectrometry systemattached to the larger scale would be used to capture spectral data;multivariate data analyses would be used to extract, from the small andlarge scale systems, features of the spectral data (e.g. principalcomponents from principal component analysis) that describe the bulk ofthe temporal variability; at the large scale, these features can then becompared with the variation observed in the same features at the smallscale, and thereby identify deviations from the typical behaviour of theprocess, for example, due to a contamination event.

According to another aspect of the invention, a system for automaticspectroscopic analysis of biological material is provided. The systemcomprises:

-   -   a plurality of sources of biological material;    -   a liquid router;    -   at least one flow cell for spectroscopy;    -   a manipulation station;    -   an interface device;        wherein:    -   the interface device is configured to receive instructions for        automatically controlling operations of the system;    -   the liquid router is configured to take a plurality of samples        of biological material from the plurality of sources, wherein        each sample of the plurality of samples is taken from a source        of the plurality of sources, and to deliver the plurality of        samples of biological material to the at least one flow cell;    -   the at least one flow cell is configured to be connected to at        least one spectrometer; and when a spectroscopic analysis of a        given sample of the plurality of samples of biological material        by the at least one spectrometer is or is predicted to be        ambiguous in that the spectroscopic analysis is affected by at        least two non-discriminable factors, the liquid router is        further configured to:

(a1) route the given sample of biological material from the at least oneflow cell to the manipulation station, wherein the manipulation stationis configured so that the given sample of biological material ismanipulated in order to impact the spectroscopic analysis; and

(b1) deliver the given manipulated sample of biological material fromthe manipulation station to the at least one flow cell; or.

the liquid router alternatively is further configured to:

(a2) take a secondary sample of biological material from the same sourceof the given sample for which the spectroscopic analysis is or ispredicted to be ambiguous;

(b2) route the secondary sample of biological material from the sourceto a manipulation station, wherein the manipulation station isconfigured so that the secondary sample of biological material ismanipulated in order to impact the spectroscopic analysis; and

(c2) deliver the manipulated secondary sample of biological materialfrom the manipulation station to the at least one flow cell.

In some examples, the system may comprise more than one manipulationstation. The system may further comprise storage stations in whichcomponents such as cleaning liquid and substances for manipulation arestored. Additionally, the system may comprise a waste station fordisposal of samples. Further, the system may comprise one or more on- orat-line reference systems.

The system is configured to autonomously and automatically perform themethod described above. In the system, the plurality of sources arecomplemented by an on-line analysis module, comprising the flow cell(s)connected to the spectrometer(s), and one or more manipulation stations.A liquid router connects the different components of the system.Accordingly, the system can autonomously perform a set of productionprocesses (e.g. cell cultivations), material manipulations and spectralcaptures.

The system may be controlled by a control computer via the interfacedevice, which receives instructions from the control computer forautomatically controlling operations of the system. In particular, theliquid router as well as potential manipulation tools may be controlledvia the interface device. The liquid router and the manipulation toolsmay comprise robotic components that perform actions on the basis ofreceived instructions. The spectrometer(s) connected to the flow cell(s)may also be controlled by the same control computer, directly orindirectly by means of a “slave” spectrometer control computer. Thecontrol computer may be in communication with the computing deviceconfigured to configured to compare results of the spectroscopicanalysis with results of the off-line reference analysis.

In particular, the steps of the method may form a protocol defining allprocedures that occur within the system, including (the variations of)the production process performed in the plurality of sources ofbiological material and the analysis step (with disambiguating step).This protocol may be written in the form of computer instructions thatcan be received by the interface device and forwarded to the componentsof the system that automatically execute the instructions without userinteraction. In particular, these may be the robotic components of theliquid outer, such as the liquid handling robot, and the manipulationtools, as well as robotic elements in the bioreactors responsible forperforming (the variations of) the production process, e.g. cellcultivation.

The invention does not only allow for automated process execution anddata acquisition but also for on-line data evaluation. The spectrum of agiven sample is automatically linked to the specific source. i.e.bioreactor, and sampling time as well to the corresponding referencevalues from the reference system, when present.

To summarize, the computer-implemented method described aboveparticularly allows for a spectroscopic analysis executable in anautomated fashion by a robotic system. The analysis is performed on-lineand ex-situ, particularly with the possibility of moving the sample backand forth between the flow cell(s) and the manipulation station(s), aswell as from and to a reference system. The automatized protocolspecifically can control production processes in the bioreactors and/orsampling procedures for spectroscopic analysis and/or referenceanalysis, including pre-processing and disambiguating manipulation ofthe samples.

In particular, the combination of process variations and manipulation ofthe samples allows a comprehensive automated calibration of thespectroscopic analysis. Indeed, the invention particularly allows forautomated multi-parallel cultivations with deliberately inducedprocess/parameter variations. Consequently, automated samplingmanipulation and spectral measurements allow for an automated root causeanalysis. As an outcome, spectral deviations can be linked toprocess/analyte changes, thus enabling process control on the basis ofspectroscopy, instead of pure monitoring of spectral deviations.

BRIEF DESCRIPTION OF THE DRAWINGS

Details of exemplary embodiments are set forth below with reference tothe exemplary drawings. Other features will be apparent from thedescription, the drawings, and from the claims. It should be understood,however, that even though embodiments are separately described, singlefeatures of different embodiments may be combined to furtherembodiments.

FIG. 1 shows a block diagram of an automated system for spectroscopicanalysis of biological material.

FIG. 2 shows another block diagram of an automated system forspectroscopic analysis of biological material.

FIG. 3 shows an exemplary workflow of a spectroscopic analysis ofbiological material.

FIG. 4 shows a block diagram of an automated system for spectroscopicanalysis of biological material in terms of controlling components.

FIG. 5 shows a block diagram of a spiking process.

FIG. 6 shows an exemplary spiking plate.

FIG. 7 shows an exemplary preparation of a spiking plate.

FIG. 8 shows a block diagram of a method for spectroscopic analysis ofbiological material.

FIGS. 9 a and 9 h show exemplary methods of sample delivery to a flowcell.

DETAILED DESCRIPTION

In the following, a detailed description of examples will be given withreference to the drawings. It should be understood that variousmodifications to the examples may be made. Unless explicitly indicatedotherwise, elements of one example may be combined and used in otherexamples to form new examples.

FIG. 1 shows a block diagram of an automated system 100 forspectroscopic analysis of biological material. The various components ofthe automated system 100 are linked among themselves via the liquidrouter 10, in the sense that the liquid router may convey liquid acrossdifferent locations in the system. The automated system 100 comprisesthe following components: a plurality of sources of biological material20, one or more flow cells 30 for spectroscopy, a manipulation station40, storage stations 50 and a waste station 60. Although a case with onemanipulation station 40 will be described, the automated system 100 maycomprise a plurality of manipulation stations 40.

FIG. 2 shows another block diagram of the automated system 100 forspectroscopic analysis of biological material, wherein a schematicspatial distribution of the components is illustrated.

The automated system 100 may be considered as comprising a bioreactormodule and an analysis module. The bioreactor module comprises theplurality of bioreactor units 20, the manipulation station 40 and thestorage stations 50, i.e. it is the part of the automated system 100 inwhich the samples are generated and manipulated. The analysis modulecomprises the flow cell(s) 30 and the waste station 60 and it is thepart in which the samples are analysed and, after the analysis,discarded. At the interface between the two modules is a sample cup 15,to which samples just taken from the bioreactors or that have undergonemanipulations already are delivered before being then routed to the flowcell(s).

The liquid router 10 connects the components in both modules and it mayassume different forms in each module. In particular, the liquid router10 may comprises a liquid handling robot capable of automated pipettingand a system of tubes and pumps. In the bioreactor module the liquidrouter 10 is in the form of the liquid handling robot and in theanalysis module the liquid router 10 is in the form of pumped lines, asshown in FIG. 2 . The liquid handling robot may comprise at least onearm that can move with at least three degrees of freedom.

The plurality of sources 20 may be multi-parallel bioreactor units, suchas those of Sartorius Ambr®. In particular, the Ambr® system maycomprise the bioreactor module as an integrated unit. As explained inthe summary, each bioreactor unit may perform a variation of aproduction process, such as a cell cultivation process.

Each of the one or more flow cells 30 is configured to contain abiological material sample and let electromagnetic radiation, such aslaser, interact with the sample. The result of the interaction isanalysed via the electromagnetic radiation spectrum by a spectrometerthat is connected to the flow cell 30 via optical fibres. Thespectroscopic analysis performed by the spectrometer providesinformation about the sample of biological material and, thus, on theprocess occurring in the bioreactor unit from which the sample has beentaken. Accordingly, the process can be monitored in real time. The oneor more flow cells 30 may be temperature-controlled.

In the simplest scenario, the liquid router 10 takes a plurality ofsamples, each from one of the sources, and delivers them to the flowcell 30 for spectroscopic analysis. This is done sequentially, i.e. theliquid router 10 takes a first sample and delivers it to the flow cell30. Once the analysis is completed, the liquid router 10 routes thefirst sample from the flow cell 30 to the waste station 60 and thendraws cleaning liquid from one of the storage stations 50 in whichcleaning liquid is stored in order to clean itself and the flow cell 30.Afterwards, the liquid router 10 repeats the same procedure with asecond sample and possibly with a third sample and so on.

In particular, the samples may be delivered to the flow cell 30 ensuringthat each sample is substantially bubble-free, thereby reducing and/oreliminating the risk of spectral artefacts e.g. caused by air.Exemplarily, the flow cell 30 may be positioned such that gravitysupports filling the flow cell and/or emptying the flow cell through theinlet and/or outlet, thereby preventing gas (e.g. air) from beingtrapped within the flow cell, particularly in an area adjacent to one ormore transparent substrates (e.g. windows) used to perform thespectroscopic measurement.

FIGS. 9 a and 9 b show two exemplary methods of sample delivery to theflow cell 30 that reduce/eliminate the presence of air. In the method ofFIG. 9 a , the liquid router 30 particularly comprises a valve, which isat least a three-way valve, positioned between the origin of the sample(e.g. one of the sources 20 and/or the sample cup 15), the flow cell 30and a further destination. e.g. the waste station 60. The flow cell 30substantially is arranged or positioned in a non-horizontal positionwith respect to the ground or gravity. The three-way valve may have afirst setting, in which it allows passage of liquid between the originof the sample and the flow cell 30, and a second setting, in which itallows passage of liquid between the flow cell 30 and the waste station60. Accordingly, the valve may be set to the first setting in order tofill the flow cell 30 with the sample and, after the spectroscopicmeasurement is performed, the valve may switch to the second setting,enabling delivery of the sample to the waste station 60. In thisexample, both the origin of the sample and the further destination areconnected with the same opening, the bottom opening, of the flow cell30.

In the method of FIG. 9 b , the origin of the sample is connected withthe top opening of the flow cell 30, while the bottom opening of theflow cell 30 is connected to an intermediate sample storage, such as atube. The sample may be delivered via the top opening of the flow cell30, with the origin of the sample being positioned substantially abovethe flow cell 30. By virtue of gravity, the sample may also at leastpartly enter the intermediate sample storage placed below the flow cell30. The flow cell 30 may be finally filled by pushing or pulling/sucking(e.g. by creating an overpressure in the intermediate sample storageand/or by means of a pump, e.g. syringe or peristaltic pump) the (partof the) sample from the intermediate sample storage back to the flowcell 30. After the spectroscopic measurement, the flow cell 30 may beemptied via the bottom opening in the intermediate sample storage andthen the sample may be transferred from the intermediate sample storageto another (e.g. final) destination, such as the waste station 60.

If there is more than one flow cell 30, the liquid router 10 deliverseach sample to the plurality of flow cells 30, before moving to thefollowing sample. In particular, the liquid router 10 may deliver thesample sequentially to each flow cell, i.e. one flow cell after theother.

More complex scenarios involve manipulation of the samples of biologicalmaterial. In one case, the liquid router 10 may deliver a sample to themanipulation station 40 before delivering it to the flow cell(s) 30. Atthe manipulation station, the sample may be modified for pre-processingpurposes and/or disambiguation purposes, as discussed in the summary. Amanipulation for pre-processing purposes modifies the sample in order tobring it in the best conditions for the spectroscopic analysis, e.g. toeliminate possible hindrances to the analysis and/or to createfavourable conditions for an uncontaminated analysis. A manipulation fordisambiguation purposes modifies the sample in order to try and impactthe spectral analysis, i.e. the spectrum, so that correlations that areanticipated to be possibly found when interpreting the spectrum may bebroken. This is also useful for calibrating a model at the basis of thespectroscopic analysis. The manipulation prior to the analysis may bedone for one purpose or both purposes. The liquid router 10 routes thesample from the manipulation station 40 to the flow cell(s) 30 and fromthere to the waste station 60, as in the simplest scenario.

In another case, the liquid router 10 may deliver the sample to themanipulation station 40 after delivering it to the flow cell(s) 30, i.e.after a first spectroscopic analysis. In this case the manipulation isdone only for disambiguation purposes when correlations were predicted,suspected or found in the spectrum of the first spectroscopic analysis,and the sample is routed again to the flow cell(s) 30 for a secondspectroscopic analysis. The sample may be then delivered to the wastestation 60 or it may undergo other manipulations before that, in aback-and-forth between the manipulation station 40 and the flow cell(s)30.

Manipulation for disambiguation purposes may be performed by taking asecondary sample from the same source of biological material 20. Theliquid router 10 may take a primary sample from a source and determinethat it is or is predicted to be ambiguous and then take a secondarysample from that same source and deliver it to the manipulation station40. The secondary sample may also undergo multiple manipulations beforeeventually ending at the waste station 60.

Of course pre-analysis and post-analysis manipulations may also becombined.

When a manipulation is performed, the liquid router 10 may draw one ormore substances, such as stock solutions, nutrients, water and others,from a corresponding storage station 50 and convey them to themanipulation station 40.

Another scenario involves an off-line reference system 70, which isshown in FIG. 1 but is not part of the automated system 100. The samplefor spectroscopic analysis may also undergo an off-line referenceanalysis at the off-line reference system, wherein the off-linereference analysis may take place simultaneously to the spectroscopicanalysis or before and after it, as explained in the summary. Inparticular, the sample may be split so that the liquid router 10 candeliver a part of it to the flow cell(s) 30 and another part to atransfer container, which is then manually carried to the off-linereference system 70. The transfer container may also be temporarilylocated in the bioreactor module, so that the liquid handling robot canreach it.

In one example, the analysis module may further comprise a pH probe,which is connected to the sample cup 15 via an alternative tube linewith respect to the one leading to the flow cell 30. The pH probe mayhave no direct role in the spectroscopic analysis. However, the pHmeasurement may be a beneficial (disambiguating) adjunct to spectroscopyor off-line reference, so the capacity to route the sample to the flowcell 30, and then back to the sample cup 15 and thence to the pH probe(as an alternative workflow to that already described) may bebeneficial.

All these scenarios may be combined, so that in one example the samplemay undergo pre-analysis and post-analysis manipulations as well as oneor more off-line reference analysis.

FIG. 3 shows an exemplary workflow of a spectroscopic analysis ofbiological material involving some of the illustrated possibilities. Inparticular, FIG. 3 shows how some steps in the workflow for subsequentsamples may be performed simultaneously in order to increase throughput.

At the beginning, the liquid handling robot takes a sample from one ofthe bioreactors 20 and places it in a transfer container, such as a wellplate. A part of the sample, or subsample, is removed from the wellplate by the liquid handler while the remainder (another subsample) istaken manually to the off-line reference system 70 for a first off-linereference analysis. The sample is then transferred via the liquidhandling robot to the sample cup 15 and from there to the flow cell 30via the pumped lines. Spectral capture occurs in the flow cell 30 and aspectroscopic analysis is performed by a spectrometer. The sample istransferred back from the analysis module to the bioreactor module, viathe pumped lines and then the liquid handling robot, and ends up in thetransfer container again. From there, the sample is manually deliveredto the off-line reference system 70 for a second off-line referenceanalysis. In the meantime, the pumped lines transfer cleaning liquid tothe flow cell 30 from a storage station 50. Concurrently, the liquidhandling robot starts to take the next sample from the bioreactors 20,which is transferred to a well on the well plate, as done before. Theliquid handling robot removes part of the sample which will be deliveredto the flow cell 30, while the pumped lines get washed and the cleaningliquid is deposited in the waste station 60. The sequence is repeateduntil all samples have been analysed.

All the steps discussed with reference to the workflow above, except forthe manual transport of the sample to the off-line reference system 70,are performed automatically, in particular the steps carried out by thecomponents of the automated system 100. These components shown in FIGS.1 and 2 are controlled by a control computer, as shown in FIG. 4 .

The central control computer 400 comprises a system control module 410for controlling the automated system 100, both the bioreactor module andthe analysis module. The automated system 100 comprises an interfacedevice for receiving controls from the central control computer 400, andthe interface device may also be configured to send process data fromthe bioreactor module to the central control computer 400. The controlsgiven to the bioreactor module and the analysis module are coordinated.

The central control computer 400 further comprises a spectrometrycontrol module 420 for controlling a spectrometer control computer 490configured to control one or more spectrometers connected via opticalfibres to the one or more flow cells 30 in the analysis module. Inaddition, the central control computer 400 comprises a spectrometry datamodule 430 for receiving spectroscopic analysis data from thespectrometer(s) via the spectrometer control computer 490.

The central control computer 400, the automated system 100 and thespectrometer control computer 490 may be part of a network.Communications between the central control computer 400 and theautomated system 100 as well as between the central control computer 400and the spectrometer control computer 490 may occur via data links, e.g.via cable media and/or wireless media.

The central control computer 400 comprises a database 440 into whichprocess data and spectroscopic analysis data are stored, together withreference data coming from the off-line reference analyses. The off-linereference system 70 may also be controlled by an off-line controlcomputer system, which may send the reference data to the centralcontrol computer 400 via the Internet. Communications between theoff-line control computer system and the central control computer 400may be secured, e.g. via Internet protocol security (IPSEC) or othersecurity protocols.

The central control computer 400 may send, exemplarily without userintervention, the spectroscopic analysis data and the reference data toa computing device comprising a data analytics module, such asSartorius-Stedim Data Analytics SIMCA, for elaboration of the data.

As mentioned with reference to FIG. 2 , the automated system 100 iscapable of manipulating samples of biological material. A particulartype of manipulation is spiking, i.e. adding a known amount of ananalyte to the sample. FIG. 5 shows a block diagram of an exemplaryspiking process performed with the aid of well plate comprising at leastthree wells. A sample is taken by the liquid router 10 from a bioreactorunit source 20 and split into three subsamples delivered respectively toa first well 510, a second well 520 and a third well 530 in a plate 500.The first subsample may be mixed with 100 uL of water, the secondsubsample may be mixed with 50 uL of water and 50 uL of glucose solutionand the third subsample may be mixed with 100 uL of glucose solution.The water and glucose solution may be retrieved by the liquid router 10from a storage station 50. The liquid router 10 routes the threesubsamples to the at least one flow cell 30 sequentially.

Aliquots may be taken from each subsample for the off-line referencesystem 70, either before spiking or after spiking. If done beforespiking, corrections based on the volume and concentration of thespiking solution may be used on the off-line reference analysis results.

FIG. 6 shows an exemplary spiking plate and FIG. 7 shows an exemplarypreparation of a spiking plate. The plate shown in FIG. 6 comprises 96wells, each containing different amounts (including null amounts) offour different disambiguating substances A, B, C, D. In particular,there are 15 different combinations and each combination is repeated sixtimes. Since each well is used only once, this plate may be used forspiking six samples coming from six sources in 15 different ways.Exemplarily, the spiking may be performed according to the standardaddition method.

Spiking plate preparation needs to take into account the potential foraccumulated inaccuracies due to liquid handling. Therefore, typically,the spiking plate would be produced by a series of liquid handlingsteps, first to put together stock solution combinations, potentiallyusing a serial dilution approach, and then to aliquot these stocksolutions to target wells on the plate. FIG. 7 shows an example thereof:plate is loaded onto system with just the Group A wells filled withstock solution. A plate is also loaded onto the system with a trough ofwater. Before the analysis starts:

-   -   the Group B wells are created by the liquid handling robot by        pipetting combinations from the Group A wells;    -   the Group D wells are created by the liquid handling robot by        pipetting combinations from the Group A wells and from the water        trough;    -   the Group C wells are created by the liquid handling robot as        aliquots from the Group A wells;    -   the Group E wells are created by the liquid handling robot as        aliquots from the Group B wells;    -   the Group F wells are created by the liquid handling robot as        aliquots from the Group D wells.

Then, during the analysis step, the wells of Groups C, E and F are usedfor spiking, with a full matrix design for four vessels. This should betreated as an illustrative example, and the exact pattern of platepreparation of spike combinations will depend on the particularexperiment to be conducted.

Different possible procedures have been discussed with reference toFIGS. 1 to 7 . A non-exhaustive overview of how these procedures may becombined in a protocol is shown in FIG. 8 . The block diagram shows amethod for spectroscopic analysis of biological material, wherein thedashed-dotted blocks and arrows indicate optional steps. The diagramrefers to a single sample of biological material and the method can berepeated for all the plurality of samples of biological material.

The method starts at S801 and it may always (i.e. for each sample)comprise steps S803, S813, S815, S817 and S819 in this order.

Before step S803, the method may comprise (not shown) performing avariation of a production process in a bioreactor unit 20.

At step S803, the sample of biological material is taken from abioreactor unit 20 by the liquid router 10, in particular by the liquidhandling robot.

At step S813 the sample is delivered by the liquid router 10 to the flowcell 30. In particular, the sample coming from the bioreactor module(i.e. from the source, in the simplest case, or from a manipulationstation 40) may be delivered by the liquid handling robot to the samplecup 15 and from the sample cup 15 via pumped lines to the flow cell 30.

At step S815, a spectroscopic analysis of the sample is exemplarilyperformed on the basis of a model by the spectrometer connected to theflow cell 30 via optical fibres. In other examples, the spectroscopicanalysis may be performed for data acquisition in order to generate amodel for it.

At step S817, once the spectroscopic analysis is completed, pumped linesof the liquid router 10 transfer the sample to the waste station 60.

At step S819, the liquid router 10 draws cleaning liquid from a storagestation 50 and washes itself and the flow cell 30.

In more complex situations, the method may further comprise additionalsteps. Specifically, the method may comprise one or more of thefollowing: pre-processing, pre-analysis disambiguating manipulation,post-analysis disambiguating manipulation, pre-analysis off-linereference analysis, simultaneous off-line reference analysis andpost-analysis off-line reference analysis.

Steps S805 and S807 cover pre-processing and pre-analysis disambiguatingmanipulation when performed before step S815. while coveringpost-analysis disambiguating manipulation when performed after stepS815.

When step S805 is performed right after S803, the sample is routed fromthe source to the manipulation station 40, where it is manipulated forpre-processing and/or disambiguation (for impacting the spectroscopicanalysis), e.g. filtered and/or spiked, at S807. When step S805 isperformed after step S815, the sample is routed from the flow cell 30,i.e. from the analysis module, back to the bioreactor module, namely tothe manipulation station 40. There, it is manipulated to impact thespectroscopic analysis at S807. Afterwards, the method moves again tostep S813, possibly going through steps S809-S811.

Step S809 covers pre-analysis off-line reference analysis, simultaneousoff-line reference analysis and post-analysis off-line referenceanalysis. In particular, when the delivery to the transfer element takesplace before the spectroscopic analysis, i.e. S809 is performed beforeS815, the sample may come from the source or from the manipulationstation 40. At the transfer element the sample is split into two parts,one carried manually to the off-line reference system 70 (not shown) andthe other taken at S811 by the liquid handling robot, which is thendelivered at S813 to the flow cell 30. The spectroscopic analysis andthe off-line reference analysis may be coordinated so as to occursimultaneously or the off-line reference analysis may be performed priorto the spectroscopic analysis.

When the delivery to the transfer element takes place after thespectroscopic analysis, i.e. S809 is performed after S815, the samplemay be routed from the flow cell 30 to the transfer element, from whichit is taken to the off-line reference system 70 for post-analysisoff-line or at-line reference analysis. Since the sample has alreadybeen used for spectroscopic analysis, there is no need for splitting itinto two parts. Further, the flow cell 30 has already been emptied.Accordingly, after S809, the method may directly move to S819.

In light of what described, it is apparent that the method is flexibleand can provide performances tailored to the specific sample.

We claim:
 1. A computer-implemented method for spectroscopic analysis ofbiological material, the method including an analysis step comprising:taking, by a liquid router, a plurality of samples of biologicalmaterial from a plurality of sources, wherein each sample of theplurality of samples is taken from a source of the plurality of sources;determining whether a spectroscopic analysis for each sample of theplurality of samples is predicted to be ambiguous in that it is affectedby at least two non-discriminable factors; if the spectroscopic analysisfor a given sample of the plurality of samples of biological material ispredicted to be ambiguous, performing a disambiguating step comprising:routing, by the liquid router, the given sample of biological materialto a manipulation station; manipulating, at the manipulation station,the given sample of biological material in order to impact thespectroscopic analysis; delivering, by the liquid router, the givenmanipulated sample of biological material from the manipulation stationto at least one flow cell; performing, by at least one spectrometerconnected to the at least one flow cell, the spectroscopic analysis ofthe given manipulated sample of biological material.
 2. The methodaccording to claim 1, wherein each source of the plurality of sources isa bioreactor.
 3. The method according to claim 1, wherein manipulatingthe given sample of biological material comprises mixing the sample ofbiological material with a disambiguating substance.
 4. The methodaccording to claim 3, wherein: manipulating further comprises dividingthe given sample of biological material in a plurality of subsamples andeach subsample is mixed with a different amount of the disambiguatingsubstance; delivering the given manipulated sample comprises deliveringthe plurality of manipulated subsamples; and performing thespectroscopic analysis of the given manipulated sample comprisesperforming the spectroscopic analysis of the plurality of manipulatedsubsamples.
 5. The method according to claim 1, wherein the analysisstep further comprises: pre-processing at least one sample of theplurality of samples of biological material prior to performing thespectroscopic analysis, in particular by at least one of: filtering,heating, normalizing pH, modifying ionic strength, diluting, metabolicinactivation.
 6. The method according to claim 1, further comprising:taking, by the liquid router, a subsample of at least one sample of theplurality of samples of biological material for a reference system;performing, by the reference system, a reference analysis of thesubsample; comparing a result of the spectroscopic analysis by thespectrometer for the at least one sample of biological material with aresult of the reference analysis.
 7. The method according to claim 1,wherein the analysis step is further performed one or more additionaltimes at different time points, the method further comprising:determining a time-based profile of results of the spectroscopicanalysis.
 8. The method according to claim 1, further comprisingperforming variations of a production process in the plurality ofsources.
 9. The method according to claim 1, wherein the steps of themethod are part of a protocol that is automatically executed.
 10. Asystem for spectroscopic analysis of biological material, the systemcomprising: a plurality of sources of biological material; a liquidrouter; at least one flow cell; a manipulation station; an interfacedevice; wherein: the interface device is configured to receiveinstructions for automatically controlling operations of the system; theliquid router is configured to take a plurality of samples of biologicalmaterial from the plurality of sources, wherein each sample of theplurality of samples is taken from a source of the plurality of sources;the at least one flow cell is configured to be connected to at least onespectrometer; and when a spectroscopic analysis of a given sample of theplurality of samples of biological material by the at least onespectrometer is predicted to be ambiguous in that the spectroscopicanalysis is affected by at least two non-discriminable factors, theliquid router is further configured to: route the given sample ofbiological material to the manipulation station, wherein themanipulation station is configured so that the given sample ofbiological material is manipulated in order to impact the spectroscopicanalysis; and deliver the given manipulated sample of biologicalmaterial from the manipulation station to the at least one flow cell.11. The system according to claim 10, wherein: the liquid router isfurther configured, prior to the spectroscopic analysis, to route atleast one sample of the plurality of samples of biological material tothe manipulation station for pre-processing; and the manipulationstation is configured such that the at least one sample of biologicalmaterial is pre-processed prior to the spectroscopic analysis, inparticular by at least one of: filtering, heating, normalizing pH,modifying ionic strength, diluting, metabolic inactivation.
 12. Thesystem according to claim 10, wherein the system is configured to beconnected to a computing device, wherein: the liquid router is furtherconfigured to take a subsample of at least one sample of the pluralityof samples of biological material for a reference system; the referencesystem is configured to perform a reference analysis of the subsample;and the computing device is configured to compare a result of thespectroscopic analysis of the at least one sample of biological materialby the at least one spectrometer with a result of the referenceanalysis.
 13. The system according to claim 11, wherein the system isconfigured to be connected to a computing device, wherein: the liquidrouter is further configured to take a subsample of at least one sampleof the plurality of samples of biological material for a referencesystem; the reference system is configured to perform a referenceanalysis of the subsample; and the computing device is configured tocompare a result of the spectroscopic analysis of the at least onesample of biological material by the at least one spectrometer with aresult of the reference analysis.
 14. The system according to claim 10,wherein the liquid router comprises a liquid handling robot thatperforms automated pipetting being operable to be controlled via theinterface device.
 15. The system according to claim 11, wherein theliquid router comprises a liquid handling robot that performs automatedpipetting being operable to be controlled via the interface device. 16.The system according to claim 12, wherein the liquid router comprises aliquid handling robot that performs automated pipetting being operableto be controlled via the interface device.
 17. A computer programproduct comprising computer-readable instructions which, when loaded andexecuted on a suitable system, perform the steps of a method accordingto claim
 1. 18. A computer program product comprising computer-readableinstructions which, when loaded and executed on a suitable system,perform the steps of a method according to claim
 2. 19. A computerprogram product comprising computer-readable instructions which, whenloaded and executed on a suitable system, perform the steps of a methodaccording to claim
 3. 20. A computer program product comprisingcomputer-readable instructions which, when loaded and executed on asuitable system, perform the steps of a method according to claim 4.